This project is focused on the aerobic and non-aerobic processes that create the peptide and protein- derived quino-cofactors, pyrroloquinoline quinone (PQQ) and trihydroxyphenylalanine quinone (TPQ). The described studies explore a cohort of reactions that are characterized by both unprecedented or highly unusual mechanisms and structure function relationships. PQQ biosynthesis is one of the least well understood among the pathways that lead to enzymatic cofactors, and each of the five conserved open reading frames that encode for essential proteins in PQQ production is being studied. These include the peptide substrate (PqqA), a likely oxygenase (PqqB,) a radical SAM enzyme (PqqE), and a protein with, as yet, no defined function (PqqD). In addition to the planned studies on isolated enzymes, the importance of protein/protein complex formation will undergo detailed investigation. These studies are expected to reveal how the PQQ pathway is able to integrate reactions that either must be anaerobic (PqqE) or require the uptake of molecular oxygen (PqqC and PqqB). PqqC is an oxidase that functions in the absence of any metal or organic cofactor, catalyzing the final step in PQQ formation via an eight-electron, eight-proton oxidation. We have developed an acid quench assay that allows us to characterize many of the chemical intermediates that occur along the PqqC reaction path. This new assay is being used in conjunction with site-specific mutagenesis, to examine the role of specific amino acid side chains (i) in generating an O2 binding/reactivity pocket, (ii) in the stepwise abstraction of eight electrons and protons from the substrate, and (iii) in the creation of a pathway that permits the abstracted protons to be "guided" toward the active site oxidant. Structural studies of the PqqC/product complex implicate a large conformational change that controls O2 reactivity, and the use of substrate itself in the intra-molecular proton transfer conduit.
The final aim of this project focuses on the biogenesis of TPQ, using an expressed, precursor yeast copper amine oxidase. In contrast to PQQ production, TPQ is formed via a self-processing mechanism. These studies use an unnatural amino acid, 4-amino-phenylalanine (p-AF) that has been successfully inserted at three key positions within the enzyme active site. The properties of p-AF, in relation to the replaced tyrosines, are uncovering important features of both cofactor biosynthesis and enzymatic catalysis.

Public Health Relevance

We are investigating post-translational processes that produce the quinocofactors, pyrroloquinoline quinone (PQQ, a bacterial growth factor) and trihydroxyphenylalanine quinone (TPQ, linked to inflammatory response(s) in mammalian vasculature and adipose tissue). Non-canonical aspects of enzyme function are highlighted in these studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039296-26
Application #
8638969
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
1988-02-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
26
Fiscal Year
2014
Total Cost
$383,425
Indirect Cost
$138,425
Name
University of California Berkeley
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Barr, Ian; Latham, John A; Iavarone, Anthony T et al. (2016) Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD. J Biol Chem 291:8877-84
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Zhang, Jianyu; Kulik, Heather J; Martinez, Todd J et al. (2015) Mediation of donor-acceptor distance in an enzymatic methyl transfer reaction. Proc Natl Acad Sci U S A 112:7954-9
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Klinman, Judith P; Bonnot, Florence (2014) Intrigues and intricacies of the biosynthetic pathways for the enzymatic quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ. Chem Rev 114:4343-65
Klema, Valerie J; Solheid, Corinne J; Klinman, Judith P et al. (2013) Structural analysis of aliphatic versus aromatic substrate specificity in a copper amine oxidase from Hansenula polymorpha. Biochemistry 52:2291-301
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